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At least 127 records · Page 7Linked to original sources
PubMed1998

EbEST: an automated tool using expressed sequence tags to delineate gene structure.

Large numbers of expressed sequence tags (ESTs) continue to fill public and private databases with partial cDNA sequences. However, using this huge amount of ESTs to facilitate gene finding in genomic sequence imposes a challenge, especially to wet-lab scientists who often have limited computing resources. In an effort to consolidate the information hidden in the vast number of ESTs into a readable and manageable format, we have developed EbEST-a program that automates the process of using ESTs to help delineate gene structure in long stretches of genomic sequence. The EbEST program consists of three functional modules-the first module separates homologous ESTs into clusters and identifies the most informative ESTs within each cluster; the second module uses the informative ESTs to perform gapped alignment and to predict the exon-intron boundary; and the third module generates text file and graphic outputs that illustrate the orientation, exonic structure, and untranslated regions (UTRs) of putative genes in the genomic sequence being analyzed. Evaluation of EbEST with 176 human genes from the ALLSEQ set indicated that it performed in-line with several existing gene finding programs, but was more tolerant to sequencing errors. Furthermore, when EbEST was challenged with query sequences that harbor more than one gene, it suffered only a slight drop in performance, whereas the performance of the other programs evaluated decreased more. EbEST may be used as a stand-alone tool to annotate human genomic sequences with EST-derived gene elements, or can be used in conjunction with computational gene-recognition programs to increase the accuracy of gene prediction. [EbBEST is available at http://EbEST.ifrc.mcw.edu]

Base Sequence↗
PubMed1990

Cloning and sequencing of cDNAs coding for the human intra-acrosomal antigen SP-10.

cDNAs coding for the intra-acrosomal protein SP-10 were cloned and characterized as a first step in understanding the expression of this antigen during spermatogenesis. Three overlapping SP-10-specific cDNAs were isolated from a human testes cDNA expression library. These cDNAs hybridized to a 1.35-kb mRNA that was present in human testes but was not found in liver or placenta. Complete sequencing of these cDNAs, designated SP-10-5, SP-10-8, and SP-10-10, produced an 1117-bp sequence containing a 265-amino acid-coding region for the SP-10 protein. Hydrophobicity plots generated from the deduced amino acid sequence showed a very hydrophobic amino terminus characteristic of a signal peptide. Sequence data showed that three different amino acid repeats occurred a total of 16 times in the central third of the SP-10 protein. Interestingly, cDNA SP-10-10 has an internal 57-base pair (19 amino acids) in-frame deletion that is not present in SP-10-5, suggesting that alternative splicing generates more than one SP-10 mRNA. The SP-10 protein appears to be a unique acrosomal protein, based on previous immunohistological data and the observation that SP-10 cDNA sequences did not show any significant homology to other sequences found in the Genbank, National Biomedical Research Foundation, or Swiss sequence banks. A recombinant SP-10 fusion protein was produced in an Escherichia coli expression vector and used to generate a polyclonal antiserum. This antiserum stained the acrosomal cap in situ and reacted with a similar set of peptides on Western blots as did a monoclonal antibody to SP-10.

Acrosome↗
PubMed1997

Genotype selection to rapidly breed congenic strains.

Congenic strains can now be constructed guided by the transmission of DNA markers. This allows not only selection for transmission of a desired, donor-derived differential region but also selection against the transmission of unwanted donor origin genomic material. The additional selection capacity should allow congenic strains to be produced in fewer generations than is possible with random backcrosses. Here, we consider modifications of a standard backcross breeding scheme to produce congenic mice by the inclusion of genotype-based selective breeding strategies. Simulation is used to evaluate the consequences of each strategy on the number of chromosomes that contain unwanted, donor-derived genetic material and the average length of this unwanted donor DNA for each backcross generation. Our prototypic strategy was to choose a single mouse to sire each generation using criteria designed to select against the transmission of chromosomes, other than the one containing the replacement genomic region, that contain any donor origin sequence at all. This chromosome elimination strategy resulted in an average of 16.4 chromosomes free of donor DNA in mice of the third backcross (N3) generation. A strategy based solely on positive selection for the replacement region required six backcross generations to achieve the same results.

Animals↗
PubMed2002

Problems associated with the identification of proteins in homologous families: the wool keratin family as a case study.

The keratin proteins from wool can be divided into two classes: the intermediate filament proteins (IFPs) and the matrix proteins. Using peptide mass spectral fingerprinting it was possible to match spots to the known theoretical sequences of some IFPs in web-based databases, as enzyme digestion generated sufficient numbers of peptides from each spot to achieve this. In contrast, it was more difficult to obtain good matches for some of the lower molecular weight matrix proteins. Relatively few peaks were generated from tryptic digests of high-sulfur proteins because of their lower molecular weight and the absence of basic residues in the first two-thirds of the sequence. Their high sequence homology also means that generally only a few of these peptides could be considered to be unique identifiers for each protein. Nevertheless, it was still possible to uniquely identify some of these proteins, while the presence of two peptides in the matrix-assisted laser desorption/ionization time-of-flight mass spectrum allowed classification of other protein spots as being members of this family. Only one major peptide peak was generated by the high-glycine tyrosine proteins (HGTPs) and there were relatively few sequences available in web-based databases, limiting their identification to one HGTP family.

Amino Acid Sequence↗
PubMed1991

Localization of the human complement component C3 binding site on the IgG heavy chain.

The location of the covalent binding site of the third component of complement (C3) on the IgG heavy chain was determined by sequence analysis of peptides generated by cyanogen bromide digestion of C3-IgG adducts. Activation of the alternative pathway by incubation of heat-aggregated human IgG1 with fresh normal human plasma formed covalent adducts of C3b-IgG. CNBr peptides of these adducts were transferred to a polyvinylidene difluoride membrane, and amino-terminal sequences were determined. A 40-kDa dipeptide containing the covalent bond was identified by labeling the free thiol group (generated during activation of the internal thioester of C3b) with iodo[1-14C]acetamide and analyzed by amino acid sequencing. The resulting double sequence suggested an adduct with NH2 termini at residue 938 (pro-C3 numbering) of C3 (75 residues NH2-terminal to the thioester) and residue 84 in the variable region of the IgG heavy chain. These results combined with results from hydroxylamine treatment (splits ester linkage between C3b and IgG) imply that this adduct peptide consists of a 22-kDa C3 fragment and an 18-kDa IgG fragment. Therefore, C3 binds covalently within the region extending from the last 20 residues of the variable region through the first 20 residues of CH2.

Amino Acid Sequence↗
PubMed1986

The nucleotide and partial amino acid sequence of toxic shock syndrome toxin-1.

The nucleotide sequence of toxic shock syndrome toxin-1 (TSST-1) has been determined. In addition, one-third of the predicted amino acid sequence was confirmed by amino acid sequence analysis of cyanogen bromide-generated TSST-1 protein fragments. The DNA sequencing results identified a 708-base pair open reading frame starting with an ATG, 7 base pairs downstream from a Shine-Dalgarno sequence, and terminating at a UAA stop codon. Amino acid analysis of the intact protein defined the NH2 terminus of the mature protein and located the cleavage point for the signal peptide (Ala/Ser). The signal peptide contained the first 40 amino acids and had characteristic structural similarities with other bacterial signal peptides. The coding sequence of the mature protein was 585 base pairs (194 amino acids) in length, and the molecular weight of the predicted protein was 22,049. This is in good agreement with the previously reported molecular weight of TSST-1 (22,000), as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. NH2-terminal amino acid sequence analysis performed on isolated TSST-1 CNBr fragments determined the position of the peptides in the TSST-1 sequence and verified the predicted amino acid sequence in those positions. Computer analyses of the amino acid sequence showed that TSST-1 has little or no sequence homology with biologically related toxins, streptococcal pyrogenic exotoxin A, and staphylococcal enterotoxins B and C.

Amino Acid Sequence↗
PubMed1993

Natural evolution of coronavirus defective-interfering RNA involves RNA recombination.

Defective-interfering (DI) RNAs of RNA viruses, in general, are generated and continue to evolve in size during serial undiluted passages of viruses. This evolution was thought to occur by independent generation of DI RNAs during virus passages and subsequent selection of new DI RNAs under new cellular conditions. Here we demonstrate that recombination between the old DI RNA and the helper viral RNA can be one of the mechanisms for natural DI RNA evolution. A mouse hepatitis virus (MHV) DI RNA, DIssE RNA, was transcribed in vitro and transfected into a mouse cell line infected with a different MHV strain (A59), which is distinguishable from the original natural helper MHV (JHM). During subsequent serial undiluted passages of the harvested virus, several novel DI RNA species were generated, while the original DIssE RNA disappeared by passage 11. cDNA cloning and sequence analysis of one of these novel DI RNAs, designated DI-2, revealed that it is composed of four discontinuous regions of the genomic sequence and is different from the structure of the original DIssE RNA. Sequence comparison among DI-2, DIssE, and helper MHV-A59 RNAs showed that DI-2 sequence is similar to DIssE in the first and second regions, but similar to the helper A59 virus in the third and last regions. Thus, this DI RNA was generated by RNA recombination between the original DIssE RNA and the helper viral RNA. These results indicate that recombination between DI RNA and helper virus RNA can be involved in the natural evolution of DI RNAs.

Animals↗
PubMedDate not supplied

Norrie disease and exudative vitreoretinopathy in families with affected female carriers.

PURPOSE: Norrie disease (ND) is a rare X-linked recessive disorder characterized by congenital blindness, which is often associated with sensorineural hearing loss and mental retardation. X-linked familial exudative vitreoretinopathy (FEVR) is a hereditary disorder characterized by an abnormality of the peripheral retina and is not associated with systemic diseases. X-linked recessive disorders generally do not affect females. Here we show that female carriers can be associated with manifestation of an X-linked disorder. METHODS: A four-generation family with an affected female, and a history of congenital blindness and hearing loss, was identified through the pro-band. A second family, with a full-term female infant, was evaluated through ophthalmic examinations and found to exhibit ocular features, such as retinal folds, retinal detachment and peripheral exudates. Peripheral blood specimens were collected from several affected and unaffected family members. DNA was extracted and analyzed by single-strand conformation polymorphism (SSCP) following polymerase chain reaction (PCR) amplification of the exons of the Norrie disease gene. The amplified products were sequenced by the dideoxy chain termination method. RESULTS: In an X-linked four-generation family, a novel missense (A118D) mutation in the third exon of the Norrie disease gene, was identified. The mutation was transmitted through three generations and cosegregated with the disease. The affected maternal grandmother and the unaffected mother carried the same mutation in one of their alleles. In an unrelated sporadic family, a heterozygous missense mutation (C96Y) was identified in the third exon of the Norrie disease gene in an affected individual. Analysis of exon-1 and 2 of the Norrie disease gene did not reveal any additional sequence alterations in these families. The mutations were not detected in the unaffected family members and the 116 normal unrelated controls, suggesting that they are likely to be the pathogenic mutations. CONCLUSIONS: The results further strengthen the proposal that X-linked disorders can occur in female carriers, due likely to an unfavorable X-inactivation.

Blindness↗
PubMed1999

Insertional tagging of at least two loci associated with resistance to adenine arabinoside in Toxoplasma gondii, and cloning of the adenosine kinase locus.

A genetic approach has been exploited to investigate adenylate salvage pathways in the protozoan parasite Toxoplasma gondii, a purine auxotroph. Using a new insertional mutagenesis vector designed to facilitate the rescue of tagged loci even when multiple plasmids integrate as a tandem array, 15 independent clonal lines resistant to the toxic nucleoside analog adenine arabinoside (AraA) were generated. Approximately two-thirds of these clones lack adenosine kinase (AK) activity. Parallel studies identified an expressed sequence tag (EST) exhibiting a small region of weak similarity to human AK, and this locus was tagged in several AK-deficient insertional mutants. Library screening yielded full-length cDNA and genomic clones. The T. gondii AK gene contains five exons spanning a approximately 3 kb locus, and the predicted coding sequence was employed to identify additional AK genes and cDNAs in the GenBank and dbEST databases. A genomic construct lacking essential coding sequence was used to create defined genetic knock-outs at the T. gondii AK locus, and AK activity was restored using a cDNA-derived minigene. Hybridization analysis of DNA from 13 AraA-resistant insertional mutants reveals three distinct classes: (i) AK-mutants tagged at the AK locus; (ii) AK- mutants not tagged at the AK locus, suggesting the possibility that another locus may be involved in regulating AK expression; and (iii) mutants with normal AK activity (potential transport mutants).

Adenosine Kinase↗
PubMed1992

A general strategy for random insertion and substitution mutagenesis: substoichiometric coupling of trinucleotide phosphoramidites.

Results from a number of recent studies suggest that amino acid insertion mutations may provide an important alternative to substitution mutations for modifying protein structures and functional activities. To facilitate the use of single-amino acid insertions, we have developed a general strategy for inducing random, in-phase codon insertions across a defined segment of a cloned gene. In brief, a mixture of blocked and protected trinucleotide phosphoramidites is coupled at substoichiometric levels after every third monomer coupling on a conventional solid-state synthesizer. From the heterogeneous mixture of oligonucleotide sequences thus generated, those oligonucleotides that have acquired a single additional codon are purified by urea/PAGE. By using equimolar amounts of GCT and GGT trinucleotides in the oligonucleotide synthesis plus standard oligonucleotide-directed mutagenesis techniques, we have induced as many as 13 different single alanine and glycine insertion mutations into the gene for staphylococcal nuclease in one experiment. On replacement of the 5'-dimethoxytrityl blocking group on the trinucleotide phosphoramidite with an acid-stable blocking group, such as levulinate or fluoren-9-ylmethoxycarbonyl (Fmoc), this same strategy of substoichiometric couplings at codon boundaries should permit the synthesis of complex pools of oligonucleotides for the introduction, with constant efficiency, of every type of amino acid substitution at each codon across a gene segment.

Base Sequence↗
PubMed2002

Sequential muscle activity and its functional role in the upper extremity and trunk during overarm throwing.

The proximal-to-distal segmental sequence has been identified in many sports activities, including baseball pitching and ball kicking. However, proximal-to-distal sequential muscle activity has not been identified. The aims of this study were to establish whether sequential muscle activity does occur and, if it does, to determine its functional role. We recorded surface electromyograms (EMGs) for 17 muscles from the upper extremity and abdomen during overarm throwing and detected the onset and peak times as indices of muscle activity. The following electromyographic properties were commonly identified in the participants. First, sequential muscle activity was observed from the scapular protractors to the shoulder horizontal flexors and from the shoulder horizontal flexors to the elbow extensor, but not from the elbow extensor to the wrist flexor or forearm pronator. Secondly, the external oblique contralateral to the throwing arm became activated before the ipsilateral external oblique. This sequence is considered to be very effective for the generation of high force and energy in the trunk. Thirdly, the ipsilateral external oblique began its activity almost at foot strike. Finally, the main activity of the rectus abdominis appeared just before the point of release.

Abdominal Muscles↗
PubMed1994

Life cycles and host-parasite relationships of Microsporidia in culicine mosquitoes.

Heterosporous (polymorphic) microsporidia in mosquitoes are characterized by intricate life cycles involving multiple spore types responsible for horizontal (per os) and vertical (transovarial) transmission. They affect two generations of the mosquito and some involve an obligate intermediate host. Heterosporous microsporidia are generally very host and tissue specific with complex developmental sequences comprised of unique stages and events. Full details on the intricate relationships between heterosporous microsporidia and their mosquito hosts have only recently been elucidated. Edhazardia aedis (Kudo, 1930) and Culicospora magna (Kudo, 1920) have developmental sequences in larvae that involve gametogony followed by plasmogamy and nuclear association to form diplokarya. These diplokaryotic stages then undergo karyogamy and form binucleate spores responsible for transovarial transmission. In the filial generation, haplosis occurs as a result of nuclear dissociation to produce uninucleate spores infectious to larval mosquitoes. Amblyospora californica (Kellen et Lipa, 1960) has similar sequences except that haplosis is by meiosis to produce spores infectious for a copepod intermediate host. A third spore type is formed in the intermediate host responsible for infection in a new generation of the mosquito host.

Animals↗
PubMed1996

Lambda clone B22 contains a 7676 bp genomic fragment of Saccharomyces cerevisiae chromosome VII spanning the VAM7-SPM2 intergenic region and containing three novel transcribed open reading frames.

A genomic clone of 7676 bp designated B22 from Saccharomyces cerevisiae has been sequenced. The 5' end matches the previously described gene, VAM7, and the 3' end matches the previously described gene, SPM2, both of which have been assigned to the left arm of chromosome VII. The intergenic region contains three transcribed open reading frames (ORFs). The first is related to an uncharacterized ORF of Bacillus subtilis and more weakly to MesJ in Escherichia coli; this is found as a single transcript of 1.1 kb by Northern blotting. The second ORF encodes a small ras-like GTPase of 222 residues with strong homology to yeast Ypt8p and to mammalian Rab11; this is found as a single transcript of 1.1 kb by Northern blotting. The third ORF generates a transcript of 1.6 kb and encodes a protein of 382 residues including a perfect match to the consensus sequence of a C2H2 zinc finger domain; it shares a strong homology with yeast Mig1p and Cre-A from Aspergillus, Emericella and E. coli. This ORF also has a striking similarity to a putative 43 kDa zinc finger protein encoded by an ORF (YEL8) immediately downstream of YPT8, raising the possibility that a region between VAM7 and SPM2 on chromosome VII arose as a duplication of the YPT8-YEL8 region of chromosome V, followed by a translocation.

Amino Acid Sequence↗
PubMed1999

Characterisation of the helicostatin peptide precursor gene from Helicoverpa armigera (Lepidoptera: Noctuidae).

The gene encoding the helicostatin peptide family was isolated from a Helicoverpa armigera genomic DNA library. The deduced precursor sequence allowed unambiguous identification of all helicostatin peptides and verified the sequences of eight peptides previously isolated. The gene consists of at least three exons and encodes a precursor of 225 amino acids that contains three blocks of tandemly arranged helicostatin peptides including seven copies of the C-terminal sequence -YXFGL followed by a single Gly residue for carboxylamidation. Complete endoproteolytic processing at all possible dibasic cleavage sites would generate the seven helicostatin octapeptides previously purified from larval extracts. If processing was not complete at the third pair of basic amino acids the octadecapeptide (helicostatin IIa) would also be released. Two novel putative helicostatin peptide sequences were identified; YSKFNFGL and ERDMHRFSFGL, both of which had the C-terminal pentapeptide -FXFGL in place of the more usual -YXFGL sequence. Comparison of the helicostatin precursor with that of the cockroaches, locust and flies revealed variation in size, sequence and organisation of the 'allatostatin' precursors across different insect orders. In situ hybridisation histochemistry established that helicostatins are expressed in neurones of the central nervous system and endocrine cells of the midgut, indicating that the helicostatins are true brain-gut peptides. Northern blot analysis identified a single transcript of 1.6 kb in mRNA from whole larvae, isolated central nervous system and gut tissue.

Amino Acid Sequence↗
PubMed2001

Real time distributed processing of multiple associated pulse pattern sequences.

A Real Time Distributed Associative Memory Artificial Neural Network (RTANN) is described. This network associates groups of pulse pattern sequences. The subsequent reoccurrence of some sequences will cause the remainder to be regenerated. Training is carried out in real time simply by feeding pattern sequences directly into the network. The connections between units incorporate a wide range of transmission delays. During training the network enhances connection weights on units where coincidences occur between input and delayed pulses. Pattern regeneration utilises the reoccurrence of coincidences between delayed pulses. The simulation of an RTANN is presented. Continuous dual pattern sequences from notional sensors monitoring the shape and colour of an object were associated directly with a third dual pattern sequence having the form 'These objects look colour'. After training the network was able to correctly generate sentences describing combinations of object and colour not encountered during training.

Artificial Intelligence↗
PubMed1993

Assessment of disparate structural features in three models of the hepatitis delta virus ribozyme.

Three models for the secondary structure of the hepatitis delta virus (HDV) antigenomic self-cleaving RNA element were tested by site-directed mutagenesis. Two models in which bases 5' to the cleavage site are paired with sequence at the 3' end of the element were both inconsistent with the data from the mutagenesis. Specifically, mutations in the 3' sequence which decrease self-cleavage activity could not be compensated by base changes in the 5' sequence as predicted by these models. The evidence was consistent with a third model in which the 3' end pairs with a portion of a loop within the ribozyme sequence to generate a pseudoknot structure. This same pairing was also required to generate higher rates of cleavage in trans with a 15-mer ribozyme, thus ruling out a proposed hammerhead-like 'axehead' model for the HDV ribozyme.

Base Sequence↗
PubMed1996

Favored use of immunoglobulin V(H)4 Genes in AIDS-associated B-cell lymphoma.

We examined the lg heavy chain variable region genes (Ig V(H) genes) expressed in biopsy specimens of 10 patients with acquired immunodeficiency syndrome (AIDS)-associated lymphoma. Eight expressed Ig V(H) genes of the V(H)4 group, indicating a bias toward expression of Ig V(H) genes of this subgroup. Sequence analyses of Ig V(H) genes isolated from any one lymphoma did not reveal evidence for intraclonal diversity. However, some lymphomas express Ig V(H) genes that apparently have undergone somatic diversification and selection. In addition, we found that the sequence encoding each examined third complementarity determining region most likely resulted from D-D fusion, a process that ordinarily contributes to the generation of a relatively small proportion of the Ig heavy chain genes expressed by normal adult B cells. The noted restriction in the use of Ig V(H) genes by AIDS-associated B-cell lymphomas suggests that antigenic stimulation contributes to lymphomagenesis in patients with AIDS.

Adult↗
PubMedDate not supplied

Plasmid-mediated resistance to third-generation cephalosporins caused by point mutations in TEM-type penicillinase genes.

Infections due to strains of Klebsiella pneumoniae, Escherichia coli, and Citrobacter freundii resistant to third-generation cephalosporins have been observed recently in France and the Federal Republic of Germany. This resistance phenotype is due to the production of new plasmid-mediated, broad-substrate-range beta-lactamases designated TEM-3 to TEM-7. DNA-DNA hybridization analysis with a probe specific for TEM-1 indicated that the corresponding genes blaT-3 to blaT-7 were variants of the structural genes for TEM-type beta-lactamases. In the present studies, a 2.5-kilobase BamHI plasmid DNA fragment encoding TEM-3 was cloned in E. coli, and the entire nucleotide sequence of blaT-3 was determined. The deduced amino acid sequence of TEM-3 differed in two positions from that of the TEM-2 enzyme: lysine (TEM-3) was substituted for glutamic acid (TEM-2) at residue 104 and serine (TEM-3) for glycine (TEM-2) at residue 238 in the numbering system of Ambler. Spontaneous mutants of TEM penicillinases with increased activity against third-generation cephalosporins were obtained in vitro by selection on cefotaxime or ceftazidime. It therefore appears that mutations in TEM-type beta-lactamases contribute to resistance to new-generation cephalosporins.

Amino Acid Sequence↗